Intestinal microbiota, diet, and the host genome interact at the intestinal epithelium (IE), a single layer of cells lining the intestinal tract. Asthe most rapidly renewing tissue in the body, precise gene regulation in response to environmental changes is necessary to maintain homeostasis. Non-coding RNAs (ncRNAs) including miRNAs, have emerged as important components of gene regulatory networks. Although gastrointestinal diseases now affect nearly 1 in 5 Americans each year, the role of ncRNAs in the IE remains largely understudied. Moreover, current information is from studies of the whole intestine, which obscure cell- type specific ncRNA signatures. We hypothesize that ncRNAs are differentially expressed across functionally distinct IE cell (IEC) types, responsive to microbial presence, and critical to IEC identity and function. A better understanding of the gene regulatory networks active in distinct cell populations is necessary for understanding normal intestinal physiology and the changes that occur during gastrointestinal diseases. This proposal breaks new ground by analyzing whole transcriptome data, including mRNA, long non- coding RNA, and small RNA, from four functionally distinct cell populations of the IE. We will isolate distinct cell populations by fluorescence activated cell sorting of IE from conventionally raised, germ-free, and conventionalized Sox9-EGFP reporter mice. This permits us to isolate intestinal epithelial stem cells, progenitors, enteroendocrine cells and differentiated lineages. Next-generation sequencing will be performed to compare whole transcriptomic profiles across cell types. We will then perform integrative analysis of mRNA and ncRNA expression data to determine which ncRNAs, including miRNAs, distinguish stem cells, progenitor or differentiated lineages and regulate the response to intestinal microbiota in each cell type. The results of this proposal will have important implications for stem cell, cancer, and intestinal biology, and may provide novel therapeutic targets in the treatment of gastrointestinal diseases.